Measurement and data-processing approaches related to enzyme reaction-based biosensors have historically been based on evaluation of non-equilibrium steady-state responses. Two limitations of such analyses include the following: (i) loss of sensitivity as the substrate concentration approaches and exceeds the corresponding Michaelis constant of the enzyme immobilized on the sensor, and (ii) adverse influences on measured values due to changes in experimental variables that influence (a) rates of chemical reactions, and (b) physical processes that control the steady state response.
Similar problems have been encountered in conventional kinetic-based methods when they are applied to enzymatic determinations of analytes in homogeneous solutions (Chen, W., et al., Analytica Chimica Acta 388:231-241, 1999). Results of such analyses generally have limited ranges of linearity and are influenced by experimental variables that affect enzyme activity. Steady-state data-analysis methods applied to enzyme reaction-based sensors are influenced by variables that affect rates of reaction and rates of mass transport. However, application of initial-rate methods using enzymes in homogenous solution (i.e., kinetic-based solution methods) tend to be influenced only by variables that affect rates of reactions provided the solutions are well-stirred.
A variety of measurement and data-processing approaches have been used in attempts to reduce or eliminate problems in homogenous solution measurement of analyte concentrations including, but not limited to, the following. Engh, et al., (Anal. Chem. 60:545, 1988), used alternative applications of a rate-based approach and showed improvement in the ruggedness of enzymatic methods but also demonstrated that the methods did little to improve the sensitivity at high concentrations of substrate. For homogenous solution analyses, a two-rate method (Wentzell, P. D., et al, Anal. Chem. 58:2851, 1986) and pseudoequilibrium methods (Meiling, G. E., et al., Anal. Chem. 50:1611, 1978; Harris, R. C., Clin. Chem. 29:2079, 1983) have demonstrated the potential to reduce dependencies on experimental variables to a similar degree as has been seen with equilibrium methods. Further, the two-rate and pseudoequilibrium methods, when used in this way, appear to maintain high sensitivity for analyte concentrations above Michaelis constants.
Two-rate and pseudoequilibrium methods (based on homogenous system methods) have been applied to enzyme-based biosensor methods to determine if these methods could be adapted to biosensors such that measurement improvements would be seen which were similar to those achieved in homogenous solution (Chen, et al., Analytica Chimica Acta 388:231-241, 1999; Wentzell, P. D., et al, Anal. Chem. 58:2851, 1986; Meiling, G. E., et al., Anal. Chem. 50:1611, 1978; Harris, R. C., Clin. Chem. 29:2079, 1983). The enzyme-based biosensor typically used in such studies consisted of an enzyme and an electron mediator immobilized on the surface of a glassy-carbon electrode (e.g., Chen, et al., Analytica Chimica Acta 388:231-241, 1999). Although some improvements in performance characteristics of the enzyme-based biosensor were observed, both methods were shown to have limitations when applied to enzyme-based biosensor data.